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PubMed2000

Cytosolic tyrosine dephosphorylation of STAT5. Potential role of SHP-2 in STAT5 regulation.

STAT5, a member of the signal transducers and activators of transcription (STATs), is important in modulating T cell functions through interleukin-2 (IL-2) receptors. Like other STAT proteins, STAT5 undergoes a rapid activation and inactivation cycle upon cytokine stimulation. Tyrosine phosphorylation and dephosphorylation are critical in regulating STAT5 activity. A number of protein tyrosine kinases have been shown to phosphorylate STAT5; however, the phosphatases responsible for STAT5 dephosphorylation remain unidentified. Using CTLL-20 as a model system, we provide evidence that tyrosine dephosphorylation of STAT5 subsequent to IL-2-induced phosphorylation occurs in the absence of STAT5 nuclear translocation and new protein synthesis. Nevertheless, down-regulation of the upstream Janus kinase activity during the deactivation cycle of IL-2-induced signaling does involve new protein synthesis. These findings point to the constitutive presence of STAT5 tyrosine phosphatase activity in the cytosolic compartment. We further demonstrate that SHP-2, but not SHP-1, directly dephosphorylates STAT5 in an in vitro tyrosine phosphatase assay with purified proteins. Furthermore, tyrosine-phosphorylated STAT5 associates with the substrate-trapping mutant (Cys --> Ser) of SHP-2 but not SHP-1. These results suggest a potential role for cytoplasmic protein-tyrosine phosphatases in directly dephosphorylating STAT proteins and in maintaining a basal steady state level of STAT activity.

Biological Transport↗
PubMed2003

PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit.

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor zeta subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor zeta subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR zeta substrate (specific activity ranging from 0.23 to 40 pmol/min/microg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-zeta. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR zeta ITAMs, with PTPH1 directly dephosphorylating zeta. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR zeta.

Binding Sites↗
PubMed1998

Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila.

The Legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaires' disease. We have recently reported the identification of a galactose/N-acetyl-D-galactosamine (Gal/GalNAc) lectin in the protozoan host Hartmannella vermiformis as a receptor for attachment and invasion by L. pneumophila (Venkataraman, C., B.J. Haack, S. Bondada, and Y.A. Kwaik. 1997. J. Exp. Med. 186:537-547). In this report, we extended our studies to the effects of bacterial attachment and invasion on the cytoskeletal proteins of H. vermiformis. We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125(FAK), paxillin, and vinculin, all of which were basally tyrosine phosphorylated in resting H. vermiformis. In addition to L. pneumophila-induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated with tyrosine dephosphorylation of paxillin, pp125(FAK), and vinculin, whereas actin was minimally affected. Inhibition of bacterial attachment to H. vermiformis by Gal or GalNAc monomers blocked bacteria-induced tyrosine dephosphorylation of detergent-insoluble proteins. In contrast, inhibition of bacterial invasion but not attachment failed to block bacteria-induced tyrosine dephosphorylation of H. vermiformis proteins. This was further supported by the observation that 10 mutants of L. pneumophila that were defective in invasion of H. vermiformis were capable of inducing tyrosine dephosphorylation of H. vermiformis proteins. Entry of L. pneumophila into H. vermiformis was predominantly mediated by noncoated receptor-mediated endocytosis (93%) but coiling phagocytosis was infrequently observed (7%). We conclude that attachment but not invasion by L. pneumophila into H. vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H. vermiformis. These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis. A model for attachment and entry of L. pneumophila into H. vermiformis is proposed.

Actins↗
PubMed2003

Protein tyrosine phosphatase 1B attenuates growth hormone-mediated JAK2-STAT signaling.

Protein tyrosine phosphatase-1B (PTP-1B) attenuates insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues located in the tyrosine kinase domain of the corresponding receptors. More recently, PTP-1B was shown to modulate the action of cytokine signaling via the nonreceptor tyrosine kinase JAK2. Transmission of the growth hormone (GH) signal also depends on JAK2, raising the possibility that PTP-1B modulates GH action. Consistent with this hypothesis, GH increased the abundance of tyrosine-phosphorylated JAK2 associated with a catalytically inactive mutant of PTP-1B. GH-induced JAK2 phosphorylation was greater in knockout (KO) than in wild-type (WT) PTP-1B embryonic fibroblasts and resulted in increased tyrosine phosphorylation of STAT3 and STAT5, while overexpression of PTP-1B reduced the GH-mediated activation of the acid-labile subunit gene. To evaluate the in vivo relevance of these observations, mice were injected with GH under fed and fasted conditions. As expected, tyrosine phosphorylation of JAK2 and STAT5 occurred readily in the livers of fed WT mice and was almost completely abolished during fasting. In contrast, resistance to the action of GH was severely impaired in the livers of fasted KO mice. These results indicate that PTP-1B regulates GH signaling by reducing the extent of JAK2 phosphorylation and suggest that PTP-1B is essential for limiting the action of GH during metabolic stress such as fasting.

Animals↗
PubMed2005

Protein tyrosine phosphatase kappa and SHP-1 are involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas.

BACKGROUND: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. METHODS: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. RESULTS: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)kappa was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. CONCLUSIONS: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPkappa and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas.

Actins↗
PubMed2003

Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.

Cell Line↗
PubMed2005

Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox.

Aberrant activation of tyrosine kinases is linked causally to human cancers. Activated Cdc42-associated kinase (Ack1), an intracellular tyrosine kinase, has primarily been studied for its signaling properties but has not been linked to specific pathologic conditions. Herein, we report that expression of activated Ack1 in LNCaP cells, while minimally increasing growth in culture, enhanced anchorage-independent growth in vitro and dramatically accelerated tumorigenesis in nude mice. Molecular chaperone heat shock protein 90beta (Hsp90beta)-bound Ack1 and treatment of cells with geldanamycin, a Hsp90 inhibitor, inhibited Ack1 kinase activity and suppressed tumorigenesis. Further, we identify the tumor suppressor WW domain containing oxidoreductase (Wwox) as an Ack1-interacting protein. Activated Ack1 tyrosine phosphorylated Wwox, leading to rapid dissociation of the Ack1-Wwox complex and concomitant Wwox polyubiquitination followed by degradation. Tyrosine phosphorylation of Wwox was critical for its degradation, as splice variant WwoxDelta5-8 that was not phosphorylated by Ack1 failed to undergo polyubiquitination and degradation. It has been reported that phosphorylation of Wwox at Tyr33 stimulated its proapoptotic activity. We observed that Y33F Wwox mutant was still tyrosine phosphorylated and polyubiquitinated by Ack1 action. Site-directed mutagenesis revealed that activated Ack1 primarily phosphorylated Wwox at Tyr287, suggesting that phosphorylation of distinct tyrosine residues activate or degrade Wwox. Primary androgen-independent prostate tumors but not benign prostate showed increased tyrosine-phosphorylated Ack1 and decreased Wwox. Taken together, these data indicate that Ack1 stimulated prostate tumorigenesis in part by negatively regulating the proapoptotic tumor suppressor, Wwox. Further, these findings suggest that Ack1 could be a novel therapeutic target for prostate cancer.

Amino Acid Sequence↗
PubMed2001

Receptor-like protein tyrosine phosphatase gamma (RPTPgamma), but not PTPzeta/RPTPbeta, inhibits nerve-growth-factor-induced neurite outgrowth in PC12D cells.

Receptor-like protein tyrosine phosphatase gamma (RPTPgamma) and PTPzeta/RPTPbeta are RPTPs which structurally resemble each other and form a distinct RPTP family. Both molecules are highly expressed in the central nervous system (CNS), though RPTPgamma is distributed also in several peripheral tissues. To date, the functional differences between RPTPgamma and PTPzeta in neuronal cells have not been made clear because their substrate and ligand molecules have not been fully elucidated. To address this issue, we established PC12D cell transfectants stably expressing rat RPTPgamma or PTPzeta and analyzed the effects on cellular response to nerve growth factor (NGF). Compared with the parent PC12D cells which extend neurites vigorously in response to NGF, the transfectants expressing RPTPgamma showed almost no neurite outgrowth. In contrast, neurite extension in PTPzeta-expressing clones on NGF treatment was the same as in parent cells. We investigated differences in tyrosine phosphorylation levels in the cellular proteins in these cells after the NGF treatment before morphological charges appeared. Despite the lack of a response, major proteins and MAP kinase in RPTPgamma-expressing PC12D cells displayed normal tyrosine phosphorylation changes on NGF treatment. However, tyrosine phosphorylation levels in the protein components purified with p13(suc1) agarose (p13(suc1) complex) from RPTPgamma-expressing cells were different from those of the control cells. (1) Tyrosine-phosphorylation levels of 140- and 117-kD proteins were significantly reduced. (2) Rapid tyrosine phosphorylation of 58-kD protein induced by NGF was absent. (3) Activities of tyrosine kinases and protein kinase C in the p13(suc1) complex were markedly reduced. We found that the p13(suc1) complex also contained cytoskeletal proteins such as MAP2 and neurofilaments, but their phosphorylation levels were not different. These results indicate that RPTPgamma and PTPzeta have different substrate specificities, and RPTPgamma inhibits NGF-induced neurite outgrowth of PC12D cells through modulation of the p13(suc1) complex.

Amino Acid Sequence↗
PubMed2006

Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases.

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events.

Animals↗
PubMed2004

Continuous association of cadherin with beta-catenin requires the non-receptor tyrosine-kinase Fer.

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by alpha- and beta-catenin. The phosphorylation state of beta-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of beta-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of beta-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated beta-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a beta-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains beta-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and beta-catenin, as well as localization of cadherin and beta-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

Animals↗
PubMed1992

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.

Amino Acid Sequence↗
PubMed1997

Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5.

The L1 family comprises transmembrane cell adhesion molecules of the immunoglobulin superfamily that play an important role in neuronal migration and axon outgrowth, fasciculation, and myelination. Consistent with a crucial role in developmental processes, mutations in L1 cause severe brain malformations. Although L1 activates intracellular signaling pathways, little is known about the membrane proximal events of L1 signaling. The cytoplasmic domains of L1 family proteins contain several conserved tyrosine residues that are potential targets for receptor tyrosine kinases. Here, we report that the L1 family protein Ng-CAM is phosphorylated on tyrosine in embryonic day 13 chicken retina. This is the first demonstration of in vivo tyrosine phosphorylation of an L1-like molecule. Because chicken embryo kinase 5 (Cek5) is a receptor tyrosine kinase expressed in neuronal processes and activated in the chicken embryonic retina, we have analyzed the possible role of Cek5 in L1 phosphorylation. The rat glioblastoma cell line B28 was stably transfected with human L1. Additional transient transfection with Cek5 cDNA led to expression of Cek5 in its tyrosine-phosphorylated, activated form. Biochemical analysis revealed that L1 is phosphorylated on tyrosine in Cek5-transfected cells but not in control transfectants. Furthermore, direct phosphorylation of the L1 cytoplasmic domain by Cek5 was demonstrated in an in vitro kinase assay. Tyrosine phosphorylation may represent a novel mechanism of signal cascade initiation through L1.

Amino Acid Sequence↗
PubMed1997

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3alpha in A431 cells.

Modulation of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA/GSK-3alpha in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 microM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA/GSK-3alpha was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA/GSK-3alpha immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA/GSK-3alpha can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA/GSK-3alpha.

Animals↗
PubMed2001

Quantitative imaging of tyrosine hydroxylase and calmodulin in the human brain.

The distributions of tyrosine hydroxylase and calmodulin in adult normal postmortem human brain were analyzed quantitatively. Consecutive coronal sections were obtained from the anterior area of the right hemisphere and were stained immunohistochemically for tyrosine hydroxylase and calmodulin. Stained sections were divided into approximately 3 million microareas at 50 microm intervals, and the immunohistochemical fluorescence intensity in each area was measured by a human brain mapping analyzer, which is a microphotometry system for analysis of the distribution of neurochemicals in a large tissue slice. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all brain regions, but its intensity varied. Relatively high levels of calmodulin were observed in brain regions with high levels of tyrosine hydroxylase, though high levels of tyrosine hydroxylase were not always observed in brain regions where high levels of calmodulin were distributed. In particular, high levels of both of tyrosine hydroxylase and calmodulin were distributed in the caudate nucleus and putamen. Previously it was shown that tyrosine hydroxylase was activated and dopamine synthesis was enhanced in the neostriatum region in mice and rats by the intracerebroventricular administration of calcium through a calmodulin-dependent system. The present results combined with these previous findings suggest that the activity of tyrosine hydroxylase in the caudate nucleus and putamen of humans may also be regulated by a calcium/calmodulin-dependent system.

Aged↗
PubMed2000

Growth inhibition and protein tyrosine phosphorylation in MCF 7 breast cancer cells by a novel K vitamin.

We have now found that the most potent, Cpd 5 [2-(2-mercaptoethanol)-3-methyl-1, 4-napthoquinone], inhibits growth of doxorubicin-resistant and doxorubicin-sensitive breast cancer cells (MCF 7r and MCF 7w) in culture. Growth inhibition by Cpd 5 was antagonized by the thiol antioxidants glutathione and cysteine, but not by catalase or superoxide dismutase, suggesting that growth inhibition is probably via conjugation of cellular thiols. In support of this, we found that Cpd 5 inhibited the activity of thiol containing cellular protein tyrosine phosphatase (PTP) enzyme, with consequent induction of various tyrosine phosphoproteins, but not serine or tyrosine phosphoproteins. The tyrosine phosphorylation was also inhibited by exogenous glutathione or cysteine and could be enhanced by depletion of cellular glutathione by BSO. This effect of Cpd 5 on protein tyrosine phosphorylation was highly selective, however. Tyrosine phosphorylation of EGF-R, Erb-B2, and ERK1/2 was increased, but not that of Insulin-R or JNK. ERK1/2 tyrosine phosphorylation and growth inhibition increased with increasing concentrations of Cpd 5. Furthermore, suppression of Cpd 5-mediated ERK1/2 phosphorylation by an ERK-kinase inhibitor antagonized growth inhibition. These results suggest a strong correlation between ERK1/2 phosphorylation by Cpd 5 and growth inhibition. This novel K-vitamin analog thus inhibits MCF 7 cell growth and induces selective protein tyrosine phosphorylation.

Antineoplastic Agents↗
PubMed2000

Tyrosine supplementation for phenylketonuria.

BACKGROUND: Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap however the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in PKU. Therefore, this review aims to assess the efficasy of tyrosine supplementation for phenylketonuria. OBJECTIVES: To assess the effects of tyrosine supplementation alongside or instead of phenylalanine restricted diet for patients with phenylketonuria who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of phenylalanine restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Trials Register which is a specialist trials register which comprises references identified from comprehensive electronic database searches, handsearching relevant journals and handsearching abstract books of conference proceedings. Additional studies were identified from handsearching the Journal of Inherited Metabolic Disease (from inception, 1978, to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references. Date of the most recent search of the Group's specialised register: November 1999. SELECTION CRITERIA: All randomised or pseudo-randomised trials investigating the use of tyrosine supplementation versus placebo in patients with phenylketonuria in addition to, or instead of, a phenylalanine restricted diet. Patients treated for maternal phenylketonuria were excluded. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed the trial eligibility, methodological quality and extracted the data. MAIN RESULTS: Two trials were included with a total of 47 patients. The blood tyrosine concentrations were significantly higher in the patients receiving tyrosine supplements than those in the placebo group (weighted mean difference 22.526, 95% Confidence interval (CI) 12.182 - 32.870). No significant differences were found between any of the other outcomes measured. REVIEWER'S CONCLUSIONS: From the available evidence no recommendations can be made about whether tyrosine supplementation should be introduced into routine clinical practice. Further randomised controlled studies are required to provide further evidence.

Dietary Supplements↗
PubMed2001

Nasal absorption of tyrosine-linked model compounds.

The objective of this research is to explore the amino acid carrier-mediated pathway for nasal uptake of tyrosine-linked model compounds. These molecules were synthesized by ether linkage between the aromatic hydroxyl group of L-tyrosine and the terminal hydroxyl group of model compounds. An enzymatically stable linkage was utilized to link the model compounds to L-tyrosine so that the loss of the compound from perfusate signals absorption only without complication from metabolism. The rat in situ nasal perfusion technique was utilized in this investigation. The analyte concentrations remaining in the nasal perfusates were quantitated by a reversed-phase high-performance liquid chromatography. All L-tyrosine-linked model compounds were absorbed across the nasal mucosa (15-50% absorbed), and the extent of nasal absorption was concentration dependent. The apparent first-order rate constants of intact conjugate loss from the nasal perfusate were calculated. The Michaelis-Menten kinetic model was applied to fit the perfusion data. The Michaelis constant, K(m), and maximum or saturation rate of compound loss from the perfusate, V(max), were calculated from Lineweaver-Burk plots [e.g., K(m) and V(max) are 0.57 mM and 0.00515 micromol/min, respectively, for one of the model compounds, O-(4-carboxyphenylmethyl)-L-tyrosine]. The absorption characteristics of all these model compounds exhibited saturable kinetics, whereas their parent compounds, possessing different octanol-water partition coefficients, showed either no absorption or transport by passive diffusion. Nasal uptake of L-tyrosine conjugates was inhibited by L-tyrosine. The results from this study indicate that nasal absorption of L-tyrosine-linked compounds is probably through an amino acid transport system. Exploitation of the amino acid transport pathway may be utilized to improve nasal absorption of poorly permeable drugs.

Administration, Intranasal↗
PubMed2002

Comparative proteome bioinformatics: identification of a whole complement of putative protein tyrosine kinases in the model flowering plant Arabidopsis thaliana.

Phosphorylation by protein tyrosine kinases is crucial to the control of growth and development of multicellular eukaryotes, including humans, and it also seems to play an important role in multicellular prokaryotes. A plant tyrosine-specific kinase has not been identified yet; hence, plants have been suggested to share with unicellular eukaryote yeast a tyrosine phosphorylation system where a limited number of stress proteins are tyrosyl-phosphorylated only by a few dual-specificity (serine/threonine and tyrosine) kinases. However, preliminary evidence obtained so far suggests that tyrosine phosphorylation in plants depends on the developmental conditions. Since sequencing of the genome of the model flowering plant Arabidopsis thaliana has been recently completed, we have performed a bioinformatic screening of the whole Arabidopsis proteome to identify a model complement of bona fide protein tyrosine kinases. In silico analyses suggest that < 4% of Arabidopsis kinases are tyrosine-specific kinases, whose gene expression has been assessed by a preliminary polymerase chain reaction screening of an Arabidopsis cDNA library. Finally, immunological evidence confirms that the number of Arabidopsis proteins specifically phosphorylated on tyrosine residues is much higher than in yeast.

Amino Acid Sequence↗